Supplemental Materials for “The association between … Supplemental Materials for “The...

Supplemental Materials for “The association between hippocampal subfield

volumes and education in cognitively normal older adults and amnestic mild

cognitive impairment patients” by Kang et al.

Supplemental Methods

1. Neuropsychological test

2. APO E genotyping

3. The definition of early and late mild cognitive impairment

Supplemental Results

1. The stepwise regression analyses

2. Mediation analyses

Supplemental Tables

1. Table S1. Non-amnestic cognitive functions of the study participants

2. Table S2. Clinical Characteristics of amnestic mild cognitive impairment subjects

Supplemental Figures

1. Figure S1. Relationship between hippocampal subfield volumes and education

(plots of simple regression)

2. Figure S2. Relationships between cognitive functions and hippocampal subfield

volumes (plots of simple regression)

3. Figure S3. Relationships between cognitive functions and education (plots of simple

regression)

.

Supplemental Methods

1. Neuropsychological evaluation

Cognitive status was assessed by neuropsychological testing at the Seoul St. Mary’s

Hospital, The Catholic University of Korea. The cognitive functions of all the subjects

were assessed with the Korean version of Consortium to Establish a Registry for

Alzheimer’s Disease (CERAD-K), including Verbal Fluency (VF), 15-item Boston Naming

Test (BNT), MMSE-K, Word List Memory (WLM), Word List Recall (WLR), Word List

Recognition (WLRc), Constructional Praxis (CP), and Constructional Recall (CR). The

CERAD is the standardized clinical and neuropsychological assessment battery for the

evaluation of patients with Alzheimer's disease. The results were reviewed by a

neuropsychologist to determine whether there was evidence of cognitive impairment.

The VF score is the number of animal names that the subject speaks for himself for one

minute. The score range of BNT is from 0 to 15 points. The score range of MMSE-K is

from 0 to 30 points. The score range of WLM is from 0 to 30 points. The score range of

WLR is from 0 to 10 points. The score range of WLR is from 0 to 10 points. The score

range of WLRc is from 0 to 10 points. The score range of CP is from 0 to 11 points. The

score range of CR is from 0 to 11 points.

2. APO E genotyping

DNA was isolated from blood using the QIAmp Blood DNA Maxi Kit protocol (Qiagen,

Valencia, CA). Genotypes for two APOE SNPs, rs429358 (E*4) and rs7412 (E*2) were

determined using TaqMan SNP genotyping assays (Applied Biosystems, Foster City,

California).

3. The definition of early and late mild cognitive impairment

(i) Late mild cognitive impairment defined by the report on memory impairment and

performance on delayed verbal recall (CERAD-K Word List Recall) below 1.5 SD of the

norm, (ii) Early mild cognitive impairment defined by the report on memory

impairment and performance on delayed verbal recall (CERAD-K Word List Recall)

between 1.0 SD and 1.5 SD below the norm.

Supplemental Results

1. The stepwise regression analyses

1.1 Relationship between education and hippocampal subfield volumes 1.1.1 Lt. parasubiculum, both groups

Result of Stepwise Backward Regression

Analysis of Variance Table

Model 1: left parasubiculum ~ age + education Model 2: left parasubiculum ~ age + gender + education + APOE genotype

Res.Df RSS Df Sum of Sq F Pr(>F)

1 65 11673.14

2 63 11406.79 2 266.35 0.74 0.4833

Finally Selected Model

Result of Multiple Regression Analysis

Beta lwr upr SE std.Beta lwr upr SE t value p

(Intercept) 117.44 72.69 162.19 22.41

5.24 < 0.001

age -0.72 -1.31 -0.12 0.30 -0.27 -0.5 -0.05 0.11 -2.41 0.019

education -0.83 -1.44 -0.22 0.31 -0.31 -0.53 -0.08 0.11 -2.70 0.009

R2=0.1601, adj.R2=0.1343, F=6.2, p=0.003, AIC =550.87 Res. Df, residual degrees of freedom; RSS, residual sum of squares; Df, degree of freedom; Sq, squares;

Pr(>F), Probability of F value.

1.1.2 Lt. presubiculum, both groups


Analysis of Variance Table Model 1: left presubiculum ~ age + education Model 2: left presubiculum ~ age + gender + education + APOE genotype


1 65 132668.1

2 63 131895.6 2 772.5 0.18 0.832




(Intercept) 553.66 402.79 704.52 75.54

7.33 < 0.001

age -3.43 -5.43 -1.43 1.00 -0.38 -0.6 -0.16 0.11 -3.43 0.001

education -2.47 -4.54 -0.40 1.04 -0.26 -0.48 -0.05 0.11 -2.38 0.02

R2=0.2027, adj.R2=0.1782, F=8.26, p < 0.001, AIC =716.15

1.1.3 Lt. subiculum, both groups


Analysis of Variance Table Model 1: left subiculum ~ age + education Model 2: left subiculum ~ age + gender + education + APOE genotype


1 65 247721.8

2 63 246508.9 2 1212.94 0.15 0.8567




(Intercept) 868.76 662.61 1074.91 103.22

8.42 < 0.001

age -5.90 -8.64 -3.17 1.37 -0.46 -0.67 -0.25 0.11 -4.31 < 0.001

education -3.28 -6.11 -0.45 1.42 -0.25 -0.46 -0.04 0.11 -2.32 0.024

R2=0.2604, adj.R2=0.2376, F=11.44, p < 0.001, AIC =758.61

1.1.4 Lt. parabiculum, aMCI group

Analysis of Variance Table

Model 1: left parasubiculum ~ age + gender + education + APOE genotype Model 2: left parasubiculum ~ age + gender + education + APOE genotype


1 30 4685.8

2 30 4685.8 0 0




(Intercept) 136.31 64.82 207.79 35.00

3.89 0.001 age -0.99 -1.95 -0.02 0.47 -0.32 -0.63 -0.02 0.15 -2.09 0.045

gender 8.63 -1.91 19.17 5.16 0.25 -0.04 0.55 0.15 1.67 0.105 education -1.21 -2.23 -0.19 0.50 -0.37 -0.67 -0.07 0.15 -2.43 0.021

APOE genotype -7.48 -17.72 2.76 5.01 -0.23 -0.52 0.07 0.15 -1.49 0.146

R2=0.3581, adj.R2=0.2725, F=4.18, p=0.008, AIC =282.72

1.1.5 Lt. parabiculum, aMCI group


Analysis of Variance Table Model 1: left subiculum ~ age + education + APOE genotype Model 2: left subiculum ~ age + gender + education + APOE genotype


1 31 120000.3

2 30 119200.9 1 799.46 0.2 0.657




(Intercept) 994.71 639.35 1350.07 174.24

5.71 < 0.001

age -7.28 -12.03 -2.53 2.33 -0.46 -0.75 -0.17 0.15 -3.13 0.004

education -5.22 -10.25 -0.20 2.47 -0.31 -0.59 -0.02 0.14 -2.12 0.042

APOE genotype -51.14 -101.23 -1.05 24.56 -0.3 -0.58 -0.02 0.14 -2.08 0.046

R2=0.3933, adj.R2=0.3346, F=6.7, p=0.001, AIC =394.22

1.2 Relationship between cognitive functions and hippocampal subfield volumes

1.2.1 CERAD-K Word List Recall, Control Group

1.2.1.1 Lt. parasubiculum, Control group


Analysis of Variance Table Model 1: left parasubiculum ~ age + APOE genotype + WLR Model 2: left parasubiculum ~ age + gender + education + APOE genotype + WLR


1 29 4219.51

2 27 4100.11 2 119.4 0.39 0.6787




(Intercept) 177.51 96.80 258.22 39.46

4.50 < 0.001

age -1.16 -2.10 -0.22 0.46 -0.46 -0.82 -0.1 0.18 -2.51 0.018

APOE genotype -9.57 -20.97 1.82 5.57 -0.27 -0.57 0.04 0.16 -1.72 0.096

WLR -6.13 -9.68 -2.57 1.74 -0.65 -1.01 -0.29 0.18 -3.53 0.001

R2=0.3242, adj.R2=0.2543, F=4.64, p=0.009, AIC =263.73

1.2.1.2 Lt. presubiculum, Control group


Analysis of Variance Table Model 1: left presubiculum ~ age + WLR Model 2: left presubiculum ~ age + gender + education + APOE genotype + WLR


1 30 51168.26

2 27 49405.91 3 1762.35 0.32 0.8101




(Intercept) 682.83 414.19 951.46 131.54

5.19 < 0.001 age -3.85 -7.01 -0.69 1.55 -0.45 -0.8 -0.1 0.18 -2.49 0.018

WLR -20.89 -32.82 -8.97 5.84 -0.64 -1 -0.29 0.18 -3.58 0.001

R2=0.3065, adj.R2=0.2602, F=6.63, p=0.004, AIC =344.08

1.2.1.3 Lt. subiculum, Control group


Analysis of Variance Table Model 1: left subiculum ~ age + WLR Model 2: left subiculum ~ age + gender + education + APOE genotype + WLR


1 30 79895.44

2 27 70849.75 3 9045.7 1.15 0.3473




(Intercept) 858.80 523.12 1194.48 164.36

5.22 < 0.001 age -4.82 -8.77 -0.88 1.93 -0.49 -0.87 -0.1 0.19 -2.50 0.018

WLR -15.72 -30.62 -0.81 7.30 -0.42 -0.8 -0.04 0.19 -2.15 0.039

R2=0.1927, adj.R2=0.1389, F=3.58, p=0.04, AIC =358.78

1.2.1.4 Rt. presubiculum, Control group


Analysis of Variance Table Model 1: right presubiculum ~ age + WLR Model 2: right presubiculum ~ age + gender + education + APOE genotype + WLR


1 30 51834.81

2 27 48226.75 3 3608.07 0.67 0.5759




(Intercept) 583.75 313.37 854.13 132.39

4.41 < 0.001

age -3.28 -6.45 -0.10 1.56 -0.42 -0.8 -0.03 0.2 -2.11 0.044

WLR -12.76 -24.76 -0.75 5.88 -0.43 -0.82 -0.04 0.2 -2.17 0.038

R2=0.1656, adj.R2=0.11, F=2.98, p=0.066, AIC =344.51

1.2.1.5 Rt. subiculum, Control group


Analysis of Variance Table Model 1: right subiculum ~ age + WLR Model 2: right subiculum ~ age + gender + education + APOE genotype + WLR


1 30 66354.60

2 27 61134.16 3 5220.44 0.77 0.5217




(Intercept) 898.75 592.84 1204.66 149.79

6.00 < 0.001 age -5.31 -8.90 -1.71 1.76 -0.57 -0.94 -0.2 0.19 -3.02 0.005

WLR -14.33 -27.92 -0.75 6.65 -0.41 -0.78 -0.04 0.19 -2.16 0.039

R2=0.2405, adj.R2=0.1899, F=4.75, p=0.016, AIC =352.66

1.2.2 CERAD-K Word List Recognition, Control Group

1.2.2.1 Lt. CA1, Control group


Analysis of Variance Table Model 1: left CA1 ~ gender + education + WLRc Model 2: left CA1 ~ age + gender + education + APOE genotype + WLRc


1 29 95537.57

2 27 92372.32 2 3165.25 0.46 0.6345




(Intercept) 290.25 124.83 455.66 80.88

3.59 0.001

gender 40.63 -7.56 88.81 23.56 0.3 -0.04 0.64 0.17 1.72 0.095

education 2.90 -0.98 6.78 1.90 0.26 -0.07 0.59 0.17 1.53 0.137 WLRc 28.73 10.37 47.09 8.98 0.48 0.19 0.78 0.15 3.20 0.003

R2=0.3644, adj.R2=0.2987, F=5.54, p=0.004, AIC =366.69



Analysis of Variance Table Model 1: left CA3 ~ WLRc Model 2: left CA3 ~ age + gender + education + APOE genotype + WLRc


1 31 14005.46

2 27 13052.57 4 952.89 0.49 0.741




(Intercept) 88.51 28.95 148.08 29.21

3.03 0.005

WLRc 12.47 5.84 19.10 3.25 0.57 0.28 0.86 0.15 3.84 0.001

R2=0.3218, adj.R2=0.2999, F=14.71, p < 0.001, AIC =299.32



Analysis of Variance Table Model 1: left CA4 ~ WLRc Model 2: left CA4 ~ age + gender + education + APOE genotype + WLRc


1 31 23578.30

2 27 21196.76 4 2381.54 0.76 0.561




(Intercept) 115.72 38.43 193.00 37.89

3.05 0.005

WLRc 15.21 6.60 23.81 4.22 0.54 0.25 0.84 0.15 3.60 0.001

R2=0.2954, adj.R2=0.2726, F=12.99, p=0.001, AIC =316.51

1.2.2.4 Lt. GC-DG, Control group


Analysis of Variance Table Model 1: left GC-DG ~ APOE genotype + WLRc Model 2: left GC-DG ~ age + gender + education + APOE genotype + WLRc


1 30 34513.86

2 27 31987.37 3 2526.49 0.71 0.554




(Intercept) 133.70 37.31 230.09 47.20

2.83 0.008

APOE genotype 22.50 -8.92 53.93 15.39 0.23 -0.08 0.53 0.16 1.46 0.154

WLRc 16.83 6.18 27.48 5.22 0.5 0.2 0.81 0.16 3.23 0.003

R2=0.2809, adj.R2=0.233, F=5.86, p=0.007, AIC =331.09

1.2.2.5 Lt. subiculum, Control group


Analysis of Variance Table Model 1: left subiculum ~ APOE genotype + WLRc Model 2: left subiculum ~ age + gender + education + APOE_genotype1 + WLRc


1 30 78738.69

2 27 73099.10 3 5639.58 0.69 0.5635




(Intercept) 272.12 126.53 417.71 71.29

3.82 0.001

APOE genotype 47.85 0.39 95.31 23.24 0.34 0.02 0.66 0.16 2.06 0.048

WLRc 16.22 0.14 32.31 7.88 0.34 0.02 0.66 0.16 2.06 0.048

R2=0.2044, adj.R2=0.1513, F=3.85, p=0.032, AIC =358.3 1.2.2.6 Lt. total hippocampus, Control group


Analysis of Variance Table Model 1: left total hippocampus ~ WLRc Model 2: left total hippocampus ~ age + gender + education + APOE genotype + WLRc


1 31 3247719

2 27 2828408 4 419310.99 1 0.4244




(Intercept) 1882.69 975.64 2789.73 444.74

4.23 < 0.001

WLRc 150.21 49.21 251.20 49.52 0.48 0.17 0.79 0.16 3.03 0.005

R2=0.2289, adj.R2=0.204, F=9.2, p=0.005, AIC =479.05

1.2.2.7 Rt. CA4, Control group


Analysis of Variance Table Model 1: right CA4 ~ gender + education + WLRc Model 2: right CA4 ~ age + gender + education + APOE genotype + WLRc


1 29 19645.39

2 27 19127.14 2 518.24 0.37 0.697




(Intercept) 161.46 86.45 236.47 36.67

4.40 < 0.001

gender 19.70 -2.15 41.55 10.68 0.34 -0.02 0.7 0.18 1.84 0.075

education 1.53 -0.22 3.29 0.86 0.32 -0.03 0.68 0.18 1.78 0.085

WLRc 9.18 0.85 17.51 4.07 0.36 0.05 0.68 0.16 2.26 0.032

R2=0.2841, adj.R2=0.21, F=3.84, p=0.02, AIC =314.49

1.2.2.8 Rt. total hippocampus, Control group


Analysis of Variance Table Model 1: right total hippocampus ~ age + WLRc Model 2: right total hippocampus ~ age + gender + education + APOE genotype + WLRc


1 30 2903657

2 27 2590957 3 312699.66 1.09 0.3717




(Intercept) 3752.70 1837.95 5667.45 937.56

4.00 < 0.001

age -19.10 -39.77 1.57 10.12 -0.3 -0.61 0.01 0.16 -1.89 0.069

WLRc 113.01 12.86 213.17 49.04 0.37 0.05 0.68 0.16 2.30 0.028

R2=0.2796, adj.R2=0.2316, F=5.82, p=0.007, AIC =477.35

1.2.3 CERAD-K Constructional Recall, aMCI Group

1.2.3.1 Lt. GC-DG, aMCI group


Analysis of Variance Table Model 1: left GC-DG ~ education + CR Model 2: left GC-DG ~ age + gender + education + APOE genotype + CR


1 32 49308.70

2 29 45777.43 3 3531.28 0.75 0.5336




(Intercept) 270.29 234.75 305.82 17.44

15.49 < 0.001

education -3.75 -6.87 -0.63 1.53 -0.38 -0.68 -0.08 0.15 -2.45 0.02

CR 7.55 1.96 13.14 2.74 0.43 0.12 0.73 0.15 2.75 0.01

R2=0.262, adj.R2=0.2159, F=5.68, p=0.008, AIC =361.09

1.2.3.2 Rt. CA4, aMCI group


Analysis of Variance Table Model 1: right CA4 ~ education + CR Model 2: right CA4 ~ age + gender + education + APOE genotype + CR


1 32 37898.05

2 29 35757.42 3 2140.64 0.58 0.6337




(Intercept) 257.78 226.63 288.93 15.29

16.86 < 0.001

education -2.50 -5.24 0.24 1.34 -0.3 -0.61 0.02 0.16 -1.86 0.072

CR 6.24 1.34 11.14 2.41 0.41 0.1 0.73 0.16 2.59 0.014

R2=0.2123, adj.R2=0.1631, F=4.31, p=0.022, AIC =351.88

1.2.3.3 Rt. GC-DG, aMCI group


Analysis of Variance Table Model 1: right GC-DG ~ gender + education + CR Model 2: right GC-DG ~ age + gender + education + APOE genotype + CR


1 31 54838.15

2 29 52643.64 2 2194.51 0.6 0.5531




(Intercept) 314.98 262.91 367.04 25.53

12.34 < 0.001 gender -24.15 -60.37 12.08 17.76 -0.22 -0.53 0.1 0.16 -1.36 0.184

education -3.41 -6.76 -0.06 1.64 -0.32 -0.62 -0.02 0.15 -2.07 0.046 CR 7.59 1.32 13.85 3.07 0.4 0.08 0.71 0.16 2.47 0.019

R2=0.295, adj.R2=0.2267, F=4.32, p=0.012, AIC =366.81

1.2.3.4 Rt. presubiculum, aMCI group


Analysis of Variance Table Model 1: right presubiculum ~ education + CR Model 2: right presubiculum ~ age + gender + education + APOE genotype + CR


1 32 91858.77

2 29 85751.19 3 6107.58 0.69 0.5664




(Intercept) 270.36 221.86 318.85 23.81

11.36 < 0.001 education -5.18 -9.44 -0.92 2.09 -0.38 -0.68 -0.08 0.15 -2.48 0.019

CR 11.16 3.53 18.79 3.75 0.45 0.16 0.75 0.15 2.98 0.005

R2=0.2825, adj.R2=0.2377, F=6.3, p=0.005, AIC =382.87

1.2.3.5 Rt. total hippocampus, aMCI group


Analysis of Variance Table Model 1: right total hippocampus ~ education + CR Model 2: right total hippocampus ~ age + gender + education + APOE genotype + CR


1 32 8509529

2 29 7828342 3 681186.97 0.84 0.4825



Beta lwr upr SE std. Beta lwr upr SE t value p

(Intercept) 3295.09 2828.32 3761.86 229.16

14.38 < 0.001

education -41.72 -82.73 -0.72 20.13 -0.33 -0.64 -0.02 0.16 -2.07 0.046

CR 93.67 20.24 167.10 36.05 0.41 0.1 0.72 0.16 2.60 0.014

R2=0.2252, adj.R2=0.1767, F=4.65, p=0.017, AIC =541.37

2. Mediation Analyses

2.1 The proposed mediator: Left GC-DG

Indirect effect of education on CERAD-K CR score

Effect Boot SE BootLLCI BootULCI

Left GC-DG .0047 .0369 -.0547 .1008

Boot, bootstrapping; SE, standard error; LLCI, lower limit of confidence interval; ULCI, upper limit of

confidence interval.

Normal Theory tests for indirect effect

Effect SE Z p

Left GC-DG .0047 .0358 .1320 .8950

SE, standard error.

2.2 The proposed mediator: Right CA4



Right CA4 .0154 .0298 -.0337 .0880




Effect SE Z p

Right CA4 .0154 .0311 .4956 .6202

SE, standard error.

2.3 The proposed mediator: Right GC-DG



Right GC-DG .0116 .0313 -.0414 .0895




Effect SE Z p

Right GC-DG .0116 .0308 .3761 .7069

SE, standard error.

2.4 The proposed mediator: Right presubiculum



Right presubiculum -.0086 .0309 -.0915 .0433




Effect SE Z p

Right presubiculum -.00086 .0308 -.2807 .7790

SE, standard error.

2.4 The proposed mediator: Right total hippocampus



Right total hippocampus -.0045 .0261 -.0351 .0732




Effect SE Z p

Right total hippocampus .0045 .0273 .1643 .8695

SE, standard error.

Supplemental Tables

Table S1. Non-amnestic cognitive functions of the study participants

Control Group

(n=39)

aMCI group

(n=38)

P value

CERAD-K VF 14.3 ± 3.7 (8-24) 12.5 ± 3.4 (7-21) 0.033*

CERAD-K BNT 11.8 ± 2.7 (5-15) 10.8 ± 2.4 (5-15) 0.093

CERAD-K CP 9.9 ± 1.5 (6-11) 9.7 ± 1.6 (5-11) 0.461

CERAD-K, the Korean version of Consortium to Establish a Registry for Alzheimer’s Disease; VF, Verbal

Fluency; BNT, 15-item Boston Naming Test; CP, Constructional Praxis. *p < 0.05

Table S2. Clinical Characteristics of amnestic mild cognitive impairment subjects

aMCI group

(n=38)

Early/late aMCI (n, %) 21 : 17 (55.3 : 44.7)

aMCI, Single/Multiple domain (n, %)

4 : 34 (10.5 : 89.5)

CERAD-K WLM Impairment (n, %)

23 (60.5)

CERAD-K WLR Impairment (n, %)

34 (89.5)

CERAD-K WLRc Impairment (n, %)

24 (63.2)

CERAD-K CR Impairment (n, %)

22 (57.9)

CERAD-K, the Korean version of Consortium to Establish a Registry for Alzheimer’s Disease; WLM, Word

List Memory; WLR, Word List Recall; WLRc, Word List Recognition; CR, Constructional Recall.

Supplemental Figures

Figure S1. Relationship between hippocampal subfield volumes and education (plots of simple regression)

(A) Relationship between education and Lt. parasubiculum in both groups

(B) Relationship between education and Lt. presubiculum in both groups

(C) Relationship between education and Lt. subiculum in both groups

(D) Relationship between education and Lt. parasubiculum in aMCI group

(E) Relationship between education and Lt. subiculum in aMCI group

Figure S2. Relationships between cognitive functions and hippocampal subfield volumes (plots of simple regression)

(A) Relationship between CERAD-K WLR and Lt. parasubiculum in control group

(B) Relationship between CERAD-K WLR and Lt. presubiculum in control group

(C) Relationship between CERAD-K WLRc and Lt. CA3 in control group

(D) Relationship between CERAD-K WLRc and Lt. CA4 in control group

(E) Relationship between CERAD-K CR and Rt. presubiculum in aMCI group

Figure S3. Relationships between cognitive functions and education (plots of simple regression)

(A) Relationship between education and MMSE-K in control group

(B) Relationship between education and CERAD-K CR in control group

(C) Relationship between education and MMSE-K in aMCI group

(D) Relationship between education and CERAD-K CR in aMCI group

CERAD-K, the Korean version of Consortium to Establish a Registry for Alzheimer’s Disease; MMSE-K, The Korean version of mini mental status examination; WLR, Word List Recall; WLRc, Word List Recognition; CR, Constructional Recall

Supplemental Materials for “The association between … Supplemental Materials for “The...

Documents

Transcript of Supplemental Materials for “The association between … Supplemental Materials for “The...